Confocal microscopy with multi-spectral encoding and system and apparatus for spectroscopically encoded confocal microscopy

ABSTRACT

A scanning confocal microscopy system and apparatus, especially useful for endoscopy with a flexible probe which is connected to the end of an optical fiber ( 9 ). The probe has a grating ( 12 ) and a lens ( 14 ) which delivers a beam of multi-spectral light having spectral components which extend in one dimension across a region of an object and which is moved to scan in another dimension. The reflected confocal spectrum is measured to provide an image of the region.

[0001] This is a continuation-in-part application of U.S. ApplicationSer. No. 09/622,971, filed Aug. 24, 2000, which is a national stageapplication of International Application No. PCT/US99/04356, filed Feb.26, 1999, claiming priority to U.S. Provisional Application No.60/076,041, filed Feb. 26, 1998, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to systems (method and apparatus)for confocal microscopy for the examination or imaging of sections of aspecimen of biological tissue, and particularly to such systems usingmulti-spectral illumination and processing of multi-spectral light.

[0003] Medical imaging technology has advanced over the last twentyyears to provide physicians with indispensable information on themacroscopic anatomy of patients. Imaging techniques such as radiography,magnetic resonance imaging, computed tomography, and ultrasoundnon-invasively allow investigation of large-scale structures in thehuman body with resolutions ranging from 100 μm to 1 mm. However, manydisease processes, such as the detection of early stages of cancer,higher resolution is necessary for proper diagnosis. In addition,clinical procedures such as screening for carcinoma and the surgicaldetection of tumor margins require higher resolution diagnostic imagingmethods.

SUMMARY OF THE INVENTION

[0004] To address these and other clinical problems in situ, anon-invasive imaging technology with a resolution that approachesstandard histopathology must be used. One promising potentialnoninvasive imaging modality is a form of light microscopy known asreflectance confocal microscopy.

[0005] Currently, the use of fast scanning confocal microscopy islimited to accessible surfaces of the skin and the eye. The reason forthis is that the only reliable methods for optical scanning must beperformed in free space. In addition, the size of these optical scannersprohibit their use in small probes such as endoscopes or catheters. Itis a feature of the invention to miniaturize the fast scanning mechanismand increase the number of medical applications of confocal microscopyto include all surfaces of the body, gynecologic applications,probe-based applications, and internal organ systems.

[0006] Multi-spectral light was proposed for use in confocal microscopy,but only for imaging vertically-spaced regions of a body underexamination. See B. Picard, U.S. Pat. No. 4,965,441, issued Oct. 25,1990. An interferometer using a grating to obtain multi-spectral lightwhich is resolved in the interferometer to obtain a spectroscopic imageis disclosed in A. Knuttal, U.S. Pat. No. 5,565,986, issued Oct. 15,1996. A lens having a color separation grating which obtains amulti-spectral light is disclosed in U.S. Pat. No. 5,600,486, issuedFeb. 4, 1997. Such multi-spectral proposals are not effective for highresolution imaging using a compact, flexible probe. A confocalmicroscope system according to this invention can be miniaturized andincorporated into a compact probe. In addition, by allowing lightdelivery through a single optical fiber, the probe may also be easilyincorporated into catheters or endoscopes. Thus, a confocal microscopein accordance with the invention allows imaging of all accessiblesurfaces of the body and increases the biomedical applications ofconfocal microscopy by an order of magnitude.

[0007] Briefly described, a confocal microscopy system embodying theinvention illuminates a region of interest in a body into which saidprobe may be inserted with a confocal spectrum extending along onedimension. Optics in said probe or physical movement of said probeenabled by attachment thereto of a flexible light conductive member(which may be an optical fiber), enables scanning of said spectrum alongone or two additional dimensions thereby providing for two or threedimensional imaging of the region. The reflected confocal spectrum maybe detected or decoded spectroscopically, preferably with a heterodynedetection mechanism which may be implemented interferometrically.

[0008] The following are hereby incorporated by reference:

[0009] Corcuff, P. and J. L. Leveque, In vivo vision of the human skinwith the tandem scanning microscope. Dermatology, 1993. 186: p. 50-54;

[0010] Rajadhyaksha, M., et al., In vivo confocal scanning lasermicroscopy of human skin: Melanin provides strong contest. J. Invest.Derm., 1995. 104: p. 946;

[0011] Webb, R. H., Scanning laser ophthalmoscope, in Noninvasivediagnostic techniques in ophthalmology, B. R. Masters, Editor. 1990,Springer-Verlag: New York; and

[0012] Tearney, G. J., R. H. Webb, and B. E. Bouma, Spectrally encodedconfocal microscopy. Optics Letters, 1998. 23(15): p. 1152-1154.

[0013] In order to image the majority of accessible epithelial tissuesin vivo three important requirements must be met. First, a focused beammust be scanned across the specimen. Second, the image acquisition timehas to be sufficiently short to prevent motion artifacts. Finally, thedevice must be small enough to be incorporated into and endoscope orcatheter. Techniques such as tandem scanning and laser scanning confocalmicroscopy have been developed address the rapid beam scanningrequirements for an in vivo confocal imaging system. However, in thesemethods, high speed scanning is obtained through the use of largemechanical devices that are not easily miniaturized. As a result, theutility of these techniques is primarily limited to the fields ofdermatology and ophthalmology. A promising new a fiber optic basedtechnique, spectrally encoded confocal microscopy (“SECM”), has recentlybeen demonstrated. This technique allows reflectance confocal microscopyto be performed through a compact probe, such as a catheter orendoscope. SECM uses wavelength division multiplexing (“WDM”) to encodeone-dimensional spatial information reflected from the sample. The fastscanning axis is replaced by a series of focused points with eachlocation being represented by a different wavelength of light. Theremittance as a function of spatial position is determined by measuringthe spectrum of the reflected light (FIG. 8). A two-dimensional image iscreated by scanning the wavelength-encoded axis by slow mechanicalmotion of the probe. Thus, endoscopic devices embodying the inventionallow SECM imaging of a variety of tissues and organs either integratedwith standard endoscopes or as stand-alone devices.

[0014] In accordance with an embodiment of the invention, a devicecapable of performing in vivo endoscopic confocal microscopy isprovided. Such a device could potentially provide physicians with a toolfor performing non-invasive subcellular diagnostic imaging in internalorgan systems. Such a modality would have significant long-term impactin its ability to enable a variety of clinical applications includingcancer screening or biopsy guidance and intraoperative tumor or othertissue identification. A device embodying the present invention couldenable in vivo endoscopic confocal microscopy imaging and potentiallyallow diagnosis of critical tissues of interest. Despite the addedcomplexity, such a device could provide access to otherwise inaccessibletissues therefore significantly enhancing the value of confocalmicroscopy as a diagnostic tool.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention will be more apparent from the following drawingswherein

[0016]FIG. 1 is a schematic diagram of a spectrally encoded confocalprobe in accordance with the invention where specific wavelengths areshown for illustrative purposes, their exact values depending on theoptical parameters of the system;

[0017]FIG. 2 is a plot of spectrally encoded light obtained by confocaldetection using direct spectral detection in accordance with thisinvention, where different wavelengths are detected by turning thespectrometer grating;

[0018]FIG. 3 is a schematic diagram showing a system embodying theinvention using a spectrometer for measurement of the spectrum, I(λ),which corresponds to reflectance from different transverse locations, x,on the specimen;

[0019]FIG. 4 is a schematic diagram of a system embodying the inventionhaving spectrally encoded confocal detection using interferencespectroscopy;

[0020] FIGS. 5A-D are schematic diagrams showing: (a) image formation;(b) translation of the optical fiber in the y direction; (c) rotation ofthe optical fiber in the forward firing mode; and (d) rotation of theoptical fiber in the side firing mode;

[0021]FIG. 6 is a schematic diagram showing cross-sectional imageformation by scanning the optical fiber or the objective lens along thez axis using a system embodying the invention;

[0022]FIG. 7 is another schematic diagram of a system embodying theinvention wherein optical zoom is achieved by moving the focus of anintermediate lens in and out of the image plan of the objective;

[0023]FIG. 8 is a diagram showing the basic principles of a spectrallyencoded confocal probe in accordance with an embodiment of theinvention;

[0024]FIGS. 9A and 9B depict: (a) a SECM forward-imaging probe; and (B)a SECM side-imaging probe embodying the invention;

[0025]FIG. 10 is a diagram illustrating the forward-imaging SECM probeof FIG. 9A;

[0026]FIG. 11 is a diagram showing an angle-imaging SECM probe inaccordance with an embodiment of the invention;

[0027]FIGS. 12A and 12B illustrate slow axis scanning for theforward-imaging probe of FIG. 9A in accordance with respectiveembodiments of the invention;

[0028]FIGS. 13A and 13B illustrate slow axis scanning for theside-imaging probe of FIG. 9B in accordance with respective embodimentsof the invention;

[0029]FIGS. 14A, 14B and 14C illustrate the focus adjustment for theforward-imaging probe of FIG. 9A in accordance with respectiveembodiments of the invention;

[0030]FIGS. 15A and 15B illustrate the focus adjustment for theside-imaging probe of FIG. 9B in accordance with respective embodimentsof the invention;

[0031]FIG. 16 is a diagram illustrating a dual prism grating prism pair(or “GRISM”) according to an embodiment of the invention;

[0032]FIG. 17 illustrates a multiple prism dispersion element embodyingthe invention;

[0033]FIGS. 18A and 18B are diagrams illustrating slow scanningmechanisms by: (a) linear transduction; and (b) rotation with cam orlever mechanism, according to respective embodiments of the invention;

[0034]FIGS. 19A and 19B illustrate the slow scanning mechanisms of FIGS.18A and 18B, respectively; and

[0035]FIGS. 20A, 20B, and 20C are diagrams illustrating a slow scanningmechanism using a circular piezoelectric bimorph in accordance with anembodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Referring now to the figures, multi-spectral encoding forconfocal microscopy uses a broad bandwidth source 10 as the input to themicroscope. In the probe 8 of the microscope, the source spectrumprovided via an optical fiber 9 is dispersed by a grating 12 and focusedby an objective lens 14 onto the sample 16. A lens 9 a is preferablydisposed between the optical fiber 9 and the grating 12 to collimate thelight from the optical fiber, as shown in FIG. 1, however, lens 9 a maybe removed. The spot for each wavelength is focused at a separateposition, x, on the sample (FIG. 1). The reflectance as a function oftransverse location is determined by measuring the reflected confocalspectrum from the sample 16 returned from probe 8.

[0037] The number of wavelengths or points that may be resolved isdetermined by: $\begin{matrix}{{\frac{\lambda}{\partial\lambda} = {m\quad N}},} & (1)\end{matrix}$

[0038] where λ is the center wavelength, ∂λ is the bandwidth of thespectrum, N is the number of lines in the grating 12 illuminated by thepolychromatic input beam 10, and m is the diffraction order. If thetotal bandwidth of the source is Δλ, the number of resolvable points, nis defined by: $\begin{matrix}{{n = \frac{\Delta\lambda}{\partial\lambda}},} & (2)\end{matrix}$

[0039] For an input source with a center wavelength of 800 nm, abandwidth of 25 nm, an input spot diameter of 5 mm, a diffractiongrating of 1800 lines/mm and a diffraction order of 1, n=281 points maybe resolved by the spectrally encoded confocal system (FIG. 2). Theparameters used in this example may be found in common, inexpensiveoptical components. The number of points may be increased by simplyincreasing the input spot diameter or the bandwidth of the source.Increasing the spot diameter increases the resultant probe diameter.Increasing the bandwidth of the source could be accomplished by using abroader bandwidth superluminescent diode, a rare earth doped fibersuperfluorescent source, or a solid state modelocked laser.

[0040] Consider next the multi-spectral process. First, consider directspectral measurement. The reflectance from the sample 16 as a functionof transverse location is determined by measuring the reflected confocalspectrum from the sample arm 18. The spectrum may be measuredefficiently by incorporating the probe 8 in the sample arm of aMichelson interferometer 20 (FIG. 3) and detecting the light transmittedthrough a high resolution spectrometer 21 at the output port 19 of theinterferometer. Thus, each wavelength measured corresponds to a separateposition, x, on the sample (FIG. 3). The advantage to this method overtraditional real time confocal microscopy is that the fast axis scanning( ˜15 kHz) may be performed external to the probe 8 by the spectrometer21 with approximately 0.1 nm spectral resolution for the parametersgiven above, well within reach of high quality spectrometers.

[0041] High sensitivity may be achieved through the use of heterodynedetection. If the reference arm 22 is modulated, such as by modulator 23with mirror 24 (FIG. 3), the interference of light from the sample arm18 and the reference arm 22 will also be modulated.

[0042] High signal-to-noise ratios may be then achieved by lock-indetection on the reference arm modulation frequency of detector 26.

[0043] Another method for measuring the spectrum is interference orFourier transform spectroscopy. This may be accomplished by inserting alinearly translating mirror 28 in the reference arm 22 and measuring thecross-correlation output 30 from the interference spectrometer due tothe interference of the reflected light from the sample and referencearms 18 and 22, respectively (FIG. 4). The advantages to this type ofspectroscopic detection include the ability to achieve higher spectralresolutions than direct detection methods, efficient use of the returnedlight, inherent modulation of the reference arm 22 by the Doppler shiftof the moving mirror 28, and the capability to extract both reflectanceand phase data from the sample 16. The ability to extract phase datafrom the sample may allow detection of refractive index as a function oftransverse position, x, which is useful to reveal the molecularcomposition of the sample as well as provide an additional source ofimage contrast other than the reflectivity of the sample specimen 16.Finally, interferometric detection has the potential to allowelimination of high order multiple scattering from the confocal signalby coherence gating.

[0044] Consider finally image formation. The multi-spectral encoding ofthe transverse location, x, allows the performance of a one-dimensionalraster scan. To obtain an image, a scan of another axis must beperformed, which is usually slower. Methods of accomplishing this slowscanning of the y axis include moving the optical fiber 9 in the ydirection (FIG. 5B), or rotating the entire probe 8 around the opticalfiber axis either in a forward scanning configuration (FIG. 5C) or aside-firing configuration (FIG. 5D). Cross-sectional images may becreated by scanning the optical fiber 9 or the objective lens 14 alongthe z axis (FIG. 6). Finally, a zoom mode may be created by scanning theoptical fiber 9 (or another lens 32 between grating 12 and objectivelens 14), in and out of the image plane of the objective lens (FIG. 7).Both linear motion along the y or z axis and rotation are easilyaccomplished in a compact probe by use of piezoelectric transducers. Asshown in FIG. 5A, signals may be received by a computer 34 fromspectroscopic detector 32 by a spectrometer (such as described inconnection with FIG. 3) or Fourier transform (such as describedconnection with FIG. 4) representing an image of a microscopic sectionof the sample, and the image displayed on a display coupled to thecomputer.

[0045] As described before, spectrally encoded confocal microscopy(“SECM”) allows reflectance confocal microscopy to be performed througha compact probe, such as a catheter or endoscope. SECM uses wavelengthdivision multiplexing (“WDM”) to encode one-dimensional spatialinformation reflected from the sample. The fast scanning axis isreplaced by a series of focused points with each location beingrepresented by a different wavelength of light. The remittance as afunction of spatial position is determined by measuring the spectrum ofthe reflected light (FIG. 8). A two-dimensional image is created byscanning the wavelength-encoded axis by slow mechanical motion of theprobe. Thus, endoscopic devices embodying the invention allow SECMimaging of a variety of tissues and organs either integrated withstandard endoscopes or as stand-alone devices.

[0046]FIG. 8 illustrates the basic optical properties and components ofSECM probe 38 in accordance with an embodiment of the invention. SECMprobe 38 includes elements similar to those of probe 8 shown in FIG. 1and is denoted by like reference numerals for such elements. Descriptionof these elements has been provided above with reference to FIG. 1 andwill not be repeated here. As shown in FIG. 8, objective lens unit 14may comprise one or more (e.g., two) lenses for focusing the sourcespectrum from fiber 9 dispersed by grating 12 onto an imaging plane atsample 16. It is noted that the imaging plane may be focused on anysurface, within any portion, and the like, of sample 16. The range ofthe source spectrum dispersed by grating 12 and focused by objectivelens unit 14 on the imaging plane (from λ_(−n) through λ₀ to λ_(+n)) mayform a field of view (“FOV”) of the SECM probe 38. The range may befocused onto a first dimension, which may extend in any direction (i.e.,a vector along the first dimension may point in said any direction),including, a longitudinal direction (or along the “z-axis” as shown inFIG. 6), a direction that is substantially transverse to thelongitudinal direction, any direction therebetween, and so forth. Forexample, the first dimension may be a non-longitudinal dimensionextending in any non-longitudinal direction (i.e., not on the “z-axis”shown in FIG. 6). It is noted, of course, that a dimension, such as thefirst dimension, may extend in two opposite directions. The range may befocused onto a straight line along the first dimension, a curved line,around a circle, around an ellipse, or onto any range of points. Thefocused range may be scanned in another direction, i.e., different fromthat of a vector along the first dimension (e.g., in a direction that istransverse to a vector along the first dimension, and the like), along asecond dimension (which may also be referred to as “slow axis”) to formthe imaging plane. The spectrum may also be scanned around an axis thatextends in another direction, i.e., different from that of a vectoralong the first dimension, to form the imaging plane.

[0047]FIG. 9A shows an example of the construction, as well as the basicmechanics, of a forward-imaging SECM probe/catheter 40 according to anembodiment of the present invention. As shown therein, forward-imagingSECM probe/catheter 40 may include a an optics and control housing 45, arotator/actuator 50, and an inner core 55, which may contain a fiberoptic element (e.g., fiber 9) that is coupled to a SECM system (e.g., asshown in FIGS. 3, 4, and 5A) at the proximal end and focuses andredirects light at the distal end (FIG. 9A). Optical components (or“distal optics”) similar to those of probe 8 or 38 may be enclosed inoptics and controls housing 45 within inner core 55. Accordingly, asdescribed above, two-dimensional imaging can be achieved either byrotating or translating the inner core 55 (and thus the opticalcomponents within) or deflecting the beam. An example of opticalcomponents, and characteristics thereof, that are specifically designedfor forward-imaging SECM probe/catheter 40 according to an embodiment ofthe invention will be described in further detail below with referenceto FIG. 10. The inner core may be enclosed in a sheath 65 that mayaccommodate a guidewire 70 as well as electrical or mechanical/pneumaticconnections to the distal optics. A transparent window 72 may beprovided to protect the optical components from moisture, dust, and soforth. FIG. 9B shows an example of the construction, as well as thebasic mechanics, of a side-imaging SECM probe/catheter 42 according toan embodiment of the present invention. As shown therein, side-imagingSECM probe/catheter 42 may include elements that are similar to those offorward-imaging SECM probe. Description of these elements will not berepeated here. However, it is noted that the imaging plane (at sample16) of side-imaging SECM probe/catheter 42 may be at an angle to theaxis of the probe/catheter 42, whereas the imaging plane offorward-imaging SECM probe/catheter 40 may be extended from the distalend of optics and controls housing 45. Thus, depending on the type ofsample 16 (e.g., the surrounding structure) to be imaged, probe/catheter40 and/or probe/catheter 42 may be used. As will be described below withreference to FIG. 11, the optical components of optics and controlshousing 45 in side-imaging SECM probe/catheter 42 may be adjustable tofocus the imaging plane to any angle from the axis of the probe.

[0048] The forward imaging design of probe/catheter 40 (FIG. 9A) maypresent a challenge of aligning the beam path with the axis of theprobe/catheter 40 in the presence of a grating that may inherentlydeflect the beam (e.g., grating 12, which may be enclosed in optics andcontrols housing 45 within inner core 55). The beam path alignment maybe achieved using a grating prism pair 75, also known as GRISM (FIG.10). As shown in FIG. 10, GRISM 75 may include a prism 76 (made of amaterial characterized by refractive index n_(p) and having an angledsurface defined by Φ), a grating 77, and materials 78 and 79characterized by refractive indexes n₁ and n₂, respectively. For thisapplication a transmission mode GRISM 75 is preferred. (FIG. 10) Whileblazed and binary gratings can be used, research has shown that thepreferred embodiment includes a holographic grating fixed to the angledprism face. The holographic grating may be of any type, including, aDickson grating, and the like. Distances f1 and f2 shown in FIG. 10 mayhave a predetermined relationship with each other and/or one or morecharacteristics/parameters of GRISM 75 (e.g., n_(p), Φ, n₁, n₂,dimensions of GRISM 80, and so forth, which may be predeterminedaccording to design and material). In accordance with an embodiment ofthe invention, the prism of GRISM 75 may be made of silicon, other highrefractive index materials, and the like. When using high refractiveindex materials, appropriate anti-reflection coatings at all refractiveindex interfaces may be used to increase transmission and avoiddeleterious back-reflections.

[0049] Thus, the optical components shown in FIG. 10, including GRISM75, may be the optical components enclosed in optics and controlshousing 45 within inner core 55 of forward-imaging probe/catheter 40shown in FIG. 9A. To accommodate for the large deflection angles, highindex of refraction materials may be required. Table 1 shows a list ofdesign parameters for some of the possible designs at differentwavelengths. The design options are and depend on the allowed tradeoffsbetween field of view, resolution and availability of gratings andprisms. TABLE 1 Possible Design Parameters for a Forward Imaging Probeat Different Wavelenghts. Design Parameters Numerical Aperture 0.9 ClearAperture (mm) 4.6 Bandwidth (nm) 110 Wavelength (nm) 632 800 1046 13001500 Grating Frequency (lines/mm) 1200 1200 1100 1000 950 Prism Index ofrefraction 2.00 2.10 2.50 2.75 2.75 Design Results Field of View (μm)328 328 325 328 327 Diffraction Limited Resolution (μm) 0.184 0.2110.391 0.565 593 Bandwidth Limited Resolution (μm) 0.309 0.391 0.5110.636 733 Axial Resolution (μm) 1.55 1.957 2.559 3.18 3.669

[0050] It is noted that the design parameters may be within a range of±5% of those listed in Table 1 above.

[0051] Another possible design allows SECM imaging at different angles.This may be preferable when imaging small lumens or complicated oruneven surfaces such as those of the mouth. As shown in FIG. 11, areflective prism or reflective GRISM 80 (for simplicity hereinafterreferred to as GRISM 80) can be used to allow complete control over theimaging angle of the device (e.g., probe/catheter 42 of FIG. 9B).Distances f1 and f2 shown in FIG. 11 may have a predeterminedrelationship with each other and/or one or morecharacteristics/parameters of GRISM 80 (e.g., angle of reflection,dimensions, and so forth, of GRISM 80 which may be predeterminedaccording to design and material). In accordance with an embodiment ofthe invention, GRISM 80 may be made of silicon, other high refractiveindex materials, and the like. When using high refractive indexmaterials, appropriate anti-reflection coatings at all refractive indexinterfaces are necessary to increase transmission and avoid deleteriousback-reflections.

[0052] To achieve two-dimensional imaging, the slow axis can be scannedin a variety of ways. One possibility is to rotate the inner core 55 ofthe probe by, say, rotator 50 to image either circular (e.g., forforward-imaging probe 40 as shown in FIG. 12A) or cylindrical sections(e.g., for side-imaging probe 42 as shown in 13A). The probe can also beconfigured to linearly translate and obtain images from planes parallelto the axis of the probe (e.g., by sliding inner core 55 using anactuator 50 as shown in FIG. 13B). Another mode of operation may be todeflect the beam using mechanical or optical techniques, including, butnot limited to, piezo-electric, electro-optic, acousto-optic,mechanical, electromagnetic or pneumatic devices 85. (FIG. 12B)

[0053] The focal plane of the probe can also be adjusted to allowvisualization of different layers within the tissue under investigation.In a forward imaging catheter (40), this can be done either by applyingvariable pressure against an elastic spacer placed in front or behindthe imaging window (FIG. 14A), by linear translating the inner core withrespect to the outside sheath and the imaging window 72 (FIG. 14B), orby rotating the inner core with threaded inner optics assembly 90against an also threaded outside sheath and imaging window 72. (FIG.14C). For a side imaging probe (42) the focal plane can be adjusted by atranslator 95 (FIG. 15A), possibly mechanical, pneumatic orpiezoelectric, or a balloon 100 external to the probe. (FIG. 15B)

[0054] Another GRISM design that appears well suited for forward imagingapplication is the symmetrical dual prism design 1600 (FIG. 16). Asshown in FIG. 16, dual prism GRISM 1600 may include prisms 1605 and 1610and a grating 1615. In accordance with an embodiment of the invention,prism 1605 may be made of a material characterized by a refractive indexn_(p) and may include an angled surface defined by Φ. Grating 1615 maybe made of a material characterized by a refractive index n_(g). Grating1615 may be a holographic grating. Dual prism GRISM 1600 may besymmetrical in that prism 1610 may also be made of a materialcharacterized by refractive index n_(p) and may also include an angledsurface defined by Φ. This allows the beam in and out of the grating1615 to be at the same angle (Littrow's angle) thus making the designvery efficient at both polarizations. One or morecharacteristics/parameters of the dual prism GRISM 1600, e.g., n_(p),n_(g), Φ, and so forth, may be predetermined according to the needs ofthe application. The air adjacent the dual prism GRISM 1600, i.e.,n_(air), may be replaced with a material having a different refractiveindex n. Grating 1615 may also be separated from prisms 1605 and 1610 bya material having a predetermined n. Different choices of prism material(e.g., silicon, other high refractive index materials, and the like)and, therefore, prism angle allow to a large extend customization of theoutput beam spread (Δθ) to match the device's requirements. To minimizebeam clipping, the total length of the dispersive optical element, whilemaximizing dispersion, high index of refraction materials may berequired for the prism. Table 2 shows a list of design parameters forsome of the possible designs at different wavelengths. The key advantageto this configuration is the ability to achieve high spectral dispersionwhile maintaining forward beam propagation. TABLE 2 Parameters for adual-prism GRISM using a Dickson holographic transmission grating andsilicon prisms. The clear aperture is 9 mm Λ lines/mm, EFL Effectivefocal length, φ grating incident angle, Δθ_(eff) - objectiveillumination angle, FOV - field of view, Δr - wavelength encodedresolution. All non-angle units are in micrometers (μm). Λ EFL φΔθ_(eff) FOV Δr Clipping 700.000 3.000e3 10.428 4.544 238.055 0.4470.212e3 3.750e3 297.568 0.558 4.500e3 357.082 0.670 5.250e3 416.5950.781 6.000e3 476.109 0.893 800.000 3.000e3 11.921 5.232 274.154 0.4500.277e3 3.750e3 342.693 0.563 4.500e3 411.231 0.675 5.250e3 479.7700.788 6.000e3 548.308 0.900 933.333 3.000e3 13.914 6.176 323.684 0.4550.376e3 3.750e3 404.605 0.569 4.500e3 485.525 0.683 5.250e3 566.4460.797 6.000e3 647.367 0.911 1120.000 3.000e3 16.708 7.558 396.324 0.4650.540e3 3.750e3 495.405 0.581 4.500e3 594.486 0.697 5.250e3 693.5670.813 6.000e3 792.648 0.929

[0055] It is noted that the design parameters may be within a range of±5% of those listed in Table 2 above. It is further noted that thepreferred embodiment may include the following parameters listed inTable 3. TABLE 3 Preferred parameters for a dual-prism GRISM using aDickson holographic transmission grating and silicon prisms. Λ EFL φΔθ_(eff) FOV Δr Clipping 1120.000 5.250e3 16.708 7.558 693.567 0.8130.540e3

[0056] It is yet further noted that the preferred design parameters maybe within a range of ±5% of those listed in Table 3 above.

[0057] Another design for forward imaging is a series of prisms (FIG.17). Although this setup suffers from less dispersive power compared tograting designs it is relatively simple and can be fabricated to a verysmall size.

[0058] Scanning the slow (y) axis can also be implemented by tilting thefiber 9 and collimator 9 a combination. This can be achieved by pivotingthe cone 105 that holds the fiber/collimator assembly by a number ofways, including push-pull (FIGS. 18A and 19A) and rotating an off-axislever (FIGS. 18B and 19B). Both these schemes can be implemented withtransducers in-line with the fiber-collimator assembly, thus notincreasing the overall diameter of the device.

[0059] Another way to scan (the fiber-collimator, a fiber or even theobjective) is by using a cylindrical piezoelectric bi-layer. Thisbimorph has the property of expanding its diameter when supplied withvoltage thus effectively scanning the object attached to the free end(FIGS. 20A, 20B, and 20C). Advantages of this design may include itssimplicity and high torque capability, but the design may require highvoltage and for small diameters the expansion may be limited by thephysical properties of piezoelectric material available today.

[0060] The systems, methods, apparatuses, and techniques of the presentinvention described above may be used for intraoperative tissueidentification. Using a probe (8, 40, or 42) embodying features of thepresent invention, a surgeon may be able to obtain information on tissuetype during an operation, thus reducing the time needed to perform theoperation and improving the outcome thereof. Time savings occurs whenduring the operation, the surgeon encounters tissue of unknown type. Anexample is identification of the parathyroid glands duringparathyroidectomy and thyroidectomy. In this type of operation, it isdifficult to identify the parathyroid (they are small and have aninconsistent anatomic location) and often other tissues are mistakenlythought to be parathyroid, resulting in accidental removal or damage ofthe parathyroid gland (in thyroidectomy surgeries) or in removal ofmuscle, fat or lymph node (in parathyroidectomy surgeries) and resultantincrease in operation time due to frozen section processing. A hand heldprobe (such as a SECM device embodying features of the presentinvention) could be used in these instances to identify the parathyroidgland and avoid incorrect surgical removal of tissue of the patient.This problem exists in other surgeries also, but is of particularimportance in head and neck surgeries due to the complex anatomy in thisanatomic region. A device embodying features of the present inventionmay be used to identify any tissue type, including, thyroid tissue,fetal tissue, and the like. Moreover, for all surgeries, thecapabilities provided by a system according to the present invention maydecrease operation time just by providing the surgeon with moreinformation prior to cutting.

[0061] From the foregoing description, it will be apparent that theinvention provides a confocal microscopy system which (a) is compact,optical fiber-based, capable of enabling confocal microscopy through aflexible catheter or endoscope; (b) is fast-scanning which takes placeexternal to the probe; (c) allows phase information to be retrieved; and(d) provides a number of resolvable points proportional to the bandwidthof the source and the beam diameter on the grating. Variations andmodifications in the herein described confocal microscopy system andprobe/catheter in accordance with the invention will undoubtedly suggestthemselves to those skilled in the art. Accordingly, the foregoingdescription should be taken as illustrative and not in a limiting sense.Thus, although preferred embodiments of the present invention andmodifications thereof have been described in detail herein, it is to beunderstood that this invention is not limited to these embodiments andmodifications, and that other modifications and variations may beeffected by one skilled in the art without departing from the spirit andscope of the invention as defined by the appended claims.

What is claimed is:
 1. A confocal microscope system which comprises aprobe movable into a body region of interest, said probe having meansfor illuminating said region with a confocal spectrum of light extendingalong one substantially transverse dimension, means for obtaining animage of the region of the specimen by moving said spectrum alonganother dimension and measuring the reflected confocal spectrum of saidlight.
 2. The system according to claim 1 wherein said probe is mountedon the end of a flexible, light-conducting member.
 3. The systemaccording to claim 2 wherein said member is an optical fiber.
 4. Thesystem according to claim 3 wherein said fiber is rotatable ortranslatable to move said probe in said another dimension.
 5. The systemaccording to claim 1 wherein said means for moving said spectrumcomprises means for moving an image plane containing said spectrumoptically or by physically moving said probe.
 6. The system according toclaim 5 wherein said probe is moved physically to scan said spectrum insaid another dimension and said probe has means for optically movingsaid image plane to scan in still another direction, thereby enabling3-D imaging.
 7. The system according to claim 1 wherein said means forobtaining said image comprises heterodyne detection means.
 8. The systemaccording to claim 7 wherein said heterodyne detection means includes aninterferometer.
 9. The system according to claim 8 wherein saidinterferometer has a sample arm terminated by said probe, a referencearm terminated by a mirror, an output arm having a spectroscopicdetector, an input arm having a source of polychromatic illumination,and a beam splitter for directing light from said source to said sampleand reference arms and directing interfering light containing saidreflected confocal spectrum into said output arm.
 10. The systemaccording to claim 9 wherein said reference arm includes means formodulating said reflected spectrum.
 11. The system according to claim 10wherein said modulating means comprising means for reciprocallyoscillating said mirror or a modulator.
 12. The system according toclaim 1 1 wherein said modulator or reciprocal oscillation is at acertain frequency, and means for lock-in operation of said detector atsaid frequency.
 13. The system according to claim 9 wherein saiddetector is a spectrometer.
 14. The system according to claim 9 whereinsaid detector includes a cross-correlator or a Fourier transformspectrometer.
 15. The system according to claim 1 wherein said probecomprises a grating and an objective which provides said confocalspectrum in an image plane of said objective.
 16. The system accordingto claim 15 wherein said probe is sufficiently small size to beinsertable into an organ internal of said body.
 17. A system forconfocally imaging tissue comprising: a source for producing light;means for producing a confocal spectrum of said light; means forfocusing said confocal spectrum in a direction into said tissue defininga first dimension and receiving returned light from said tissue, inwhich said confocal spectrum producing means is capable of providing aconfocal spectrum which when focused by said focusing means extendsalong a second dimension in said tissue different from the firstdimension; and means for detecting said returned light in accordancewith spectrum of said returned light to provide an image representingsaid tissue.
 18. The system according to claim 17 further comprisingmeans for scanning said confocal spectrum in at least one dimension withrespect to said tissue.
 19. The system according to claim 17 wherein atleast said producing means and said focusing and receiving means arelocated is a probe capable of insertion in a body.
 20. The systemaccording to claim 17 further comprising an optical fiber which providessaid light from said source to said producing means, and provides saidreturned light from said focusing and receiving means to said detectingmeans.
 21. The system according to claim 17 wherein said producing meansand focusing means are provided by more than one optical element. 22.The system according to claim 17 wherein said detecting means comprisesat least a spectrometer.
 23. The system according to claim 22 furthercomprising interferometric means for enabling said detecting means. 24.The system according to claim 17 wherein said light is polychromatic,said focusing means provides for focusing said confocal spectrum intosaid tissue along multiple positions in the tissue encoded in accordancewith characteristics of the polychromatic light and said confocalspectrum producing means, and said detecting means spectroscopicallydetects said returned light to provide an image of a section of thetissue in accordance with the encoded positions of the confocal spectrumfocused in the tissue.
 25. The system according to claim 17 wherein saidsecond dimension is substantially transverse with respect to said firstdimension.
 26. A method for confocally imaging tissue comprising thesteps of: providing a source of polychromatic light; producing aconfocal spectrum of said light with the aid of a diffractive element;focusing said confocal spectrum into said tissue along multiplesubstantially transverse positions in the tissue encoded in accordancewith characteristics of the polychromatic light and said diffractiveelement; receiving returned light from the tissue; and spectroscopicallydetecting said returned light and producing an image of a section of thetissue in accordance with the encoded positions of the confocal spectrumfocused in the tissue.
 27. A system for imaging tissue comprising: adiffractive element capable of providing illumination of one or morewavelengths along a first dimension; and a lens which focuses saidillumination in a direction into said tissue along a second dimensiondifferent from said first dimension, and said lens receives returnedillumination from said tissue representative of one or more locations insaid tissue in accordance with said one or more wavelengths.
 28. Thesystem according to claim 27 further comprising a probe comprising atleast said lens and said diffractive element.
 29. The system accordingto claim 27 wherein said first dimension is substantially transversewith respect to said second dimension.
 30. The system according to claim27 wherein said lens focuses said illumination into one or more spots inthe tissue at said one or more locations in accordance with said one ormore wavelengths.
 31. The system according to claim 27 furthercomprising means for scanning said tissue with said illumination focusedby said lens.
 32. The system according to claim 31 further comprisingmeans for detecting said returned light to provide an image of saidtissue representative of said region of said tissue.
 33. An apparatusmovable into a body region of interest for use with a confocalmicroscope system, said apparatus comprising: means for illuminating thebody region with a confocal spectrum of light extending along onenon-longitudinal dimension; and means for obtaining an image of the bodyregion by moving said spectrum along another dimension and measuring thereflected confocal spectrum of said light.
 34. An apparatus forconfocally imaging tissue, comprising: an input for receiving light froma light source; a light dispersing unit connected to the input forproducing a confocal spectrum of said light; a focusing unit operable tofocus said confocal spectrum at the tissue along a non-longitudinaldimension; and a light detecting unit operable to detect returned lightfrom the tissue in accordance with a spectrum of said returned light toprovide an image representing the tissue.
 35. The apparatus of claim 34,wherein said input comprises an optical fiber for transmitting saidlight.
 36. The apparatus of claim 34, wherein said light dispersing unitcomprises a grating.
 37. The apparatus of claim 34, wherein said lightdispersing unit comprises a light deflecting unit for deflecting saidconfocal spectrum to said dimension.
 38. The apparatus of claim 37,wherein said light deflecting unit comprises a prism.
 39. The apparatusof claim 38, wherein said light dispersing unit comprises a grating. 40.The apparatus of claim 39, wherein said grating is a holographicgrating.
 41. The apparatus of claim 39, wherein said grating is fixed toan angled face of said prism.
 42. The apparatus of claim 38, whereinsaid prism is a reflective prism.
 43. The apparatus of claim 37, whereinsaid light deflecting unit comprises a grating prism pair.
 44. Theapparatus of claim 43, wherein said light deflecting unit comprises adual prism grating prism pair.
 45. The apparatus of claim 34, furthercomprising an adjustment unit operable to adjust said confocal spectrumat the tissue.
 46. The apparatus of claim 45, wherein said adjustmentunit moves said confocal spectrum in another direction along a seconddimension.
 47. The apparatus of claim 45, wherein said adjustment unitadjusts said focusing unit.
 48. The apparatus of claim 45, wherein saidadjustment unit comprises one or more of a rotator, an actuator, a beamdeflector, and a balloon.
 49. The apparatus of claim 45, wherein saidadjustment unit adjusts said input.
 50. The apparatus of claim 49,wherein said input comprises a fiber/collimator assembly.
 51. Theapparatus of claim 50, wherein said adjustment unit comprises one ormore of a linear transduction mechanism, a rotational cam mechanism, anda rotational lever mechanism for moving said fiber/collimator assembly.52. The apparatus of claim 45, wherein said adjustment unit comprises acircular piezoelectric bimorph.
 53. The apparatus of claim 45, whereinsaid adjustment unit moves said confocal spectrum around an axis thatextends in another direction.
 54. A system for confocally imaging tissuecomprising: a light source operable to produce light; a light dispersingunit connected to the light source for producing a confocal spectrum ofsaid light; a focusing unit operable to focus said confocal spectrum atthe tissue along a non-longitudinal dimension; and a light detectingunit operable to detect returned light from the tissue in accordancewith a spectrum of said returned light to provide an image representingthe tissue.
 55. A light deflecting unit for use in an optical apparatus,comprising: a first prism having a first surface and a second surface,said second surface forming a first predetermined angle with said firstsurface; a second prism having a third surface and a fourth surface,said third surface facing said first surface of said first prism andsaid fourth surface forming a second predetermined angle with said thirdsurface; and a grating disposed between said first surface of said firstprism and said third surface of said second prism, whereby light enterssaid second surface of said first prism at approximately the same angleas a confocal spectrum of said light exits said fourth surface of saidsecond prism.
 56. The light deflecting unit of claim 55, wherein one ofsaid first prism and said second prism is made of a high refractiveindex material.
 57. The light deflecting unit of claim 56, wherein saidhigh refractive index material is silicon.
 58. The light deflecting unitof claim 55, wherein said grating is a holographic grating.
 59. Thelight deflecting unit of claim 55, wherein said first predeterminedangle is equal to said second predetermined angle.
 60. The lightdeflecting unit of claim 55, wherein all refractive index interfaces arecoated with an anti-reflection coating.
 61. A confocal microscope systemwhich comprises a probe movable into a body region of interest, saidprobe having means for illuminating said region with a confocal spectrumof light extending along one non-longitudinal dimension, means forobtaining an image of the region of the specimen by moving said spectrumalong another dimension and measuring the reflected confocal spectrum ofsaid light.
 62. An apparatus movable into a body region of interest foruse with a confocal microscope system, said apparatus comprising: anillumination unit operable to illuminate the body region with a confocalspectrum of light extending along one non-longitudinal dimension; and animage detecting unit coupled to said illumination unit, said imagedetecting unit operable to obtain an image of the body region by movingsaid spectrum along another dimension and measuring the reflectedconfocal spectrum of said light.
 63. A method of identifying one or moretargets for an operation in tissue, comprising using the systemaccording to claim 27, and identifying the one or more targets inaccordance with the returned illumination.
 64. The method of claim 63,wherein the tissue is thyroid tissue.
 65. The method of claim 63,wherein the tissue is fetal tissue.
 66. A method of identifying one ormore targets for an operation in tissue, comprising using the apparatusaccording to claim 34, and identifying the one or more targets inaccordance with the image representing the tissue.
 67. The method ofclaim 66, wherein the tissue is thyroid tissue.
 68. The method of claim66, wherein the tissue is fetal tissue.
 69. A method of identifying oneor more targets for an operation in tissue, comprising using theapparatus according to claim 54, and identifying the one or more targetsin accordance with the image representing the tissue.
 70. The method ofclaim 69, wherein the tissue is thyroid tissue.
 71. The method of claim69, wherein the tissue is fetal tissue.
 72. A method of identifying oneor more targets for an operation in a body region, comprising using theapparatus according to claim 62, and identifying the one or more targetsin accordance with the image of the body region.
 73. The method of claim72, wherein the body region comprises thyroid tissue.
 74. The method ofclaim 72, wherein the body region comprises fetal tissue.